The process of transcription is essential for all living cells. Transcription is accomplished in four steps: promoter binding, RNA chain initiation, RNA transcript elongation, and RNA transcript termination. Regulation of transcription can occur at each of these steps. Recent studies suggest that transcript elongation by RNA polymerase II is one of the major targets of gene regulation. It has been established that regulation of gene expression at the level of transcript elongation involves factors interacting with histones. These factors facilitate transient displacement of histones from chromatin during transcript elongation and modify the rate of transcription through chromatin. The long-term research goal of this proposal is to understand the mechanism and the regulation of transcript elongation by eukaryotic RNA polymerases in chromatin. When eukaryotic genes are activated for transcription, their chromatin structure changes to accommodate transcription factors and to allow efficient transcription by RNA polymerases. However, for many genes there is evidence that transcribed regions are covered with nucleosomes. This raises the questions: 1. How do polymerases transcribe through the nucleosome barrier? 2. How do the factors interacting with histones change the rate of transcript elongation? These questions will be addressed in a highly purified transcription system in vitro. We will analyze transcription of homogeneous and well-defined mono- and polynucleosomal chromatin templates using biochemical, fluorescent, molecular genetic and single-molecule techniques. Our experiments will be focused on analysis of eukaryotic RNA polymerase II. The specific aims are: 1. To test whether transient displacement of histones from nucleosome is the rate-limiting step during transcript elongation. 2. To determine whether elongation factors such as FACT facilitate transcription through chromatin by interacting with histones within polymerase-engaged nucleosomes. The discovery that some elongation factors play important roles in oncogenesis underscores the potential clinical significance of analysis of the mechanism of transcript elongation through chromatin.